Purification of Calcitonin-Like Peptides from Rat Brain and Pituitary*

Abstract

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from SpragueDawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was Nterminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptormediated functions. (Endocrinology 139: [982][983][984][985][986][987][988][989][990][991][992] 1998)

Side-Chain Lactam-Bridge Conformational Constraints Differentiate the Activities of Salmon and Human Calcitonins and Reveal a New Design Concept for Potent Calcitonin Analogues

“…The primary structures of calcitonins from a number of species are known, and these show variations in amino acid sequence throughout most of their length. 4 Nevertheless, a number of structural features are conserved that appear to be important determinants of receptor recognition and function. These include a highly conserved N-terminal disulfide loop linking cysteine residues in positions 1 and 7, a potential amphiphilic R-helical structure in residues 8 through 22, 5 and a C-terminal amide.…”

A synthetic peptide derived from mouse pituitary calcitonin cDNA sequence exhibits potent inhibition of prolactin secretion and prolactin mRNA abundance in primary mouse pituitary cells

“…Our previous studies have demonstrated that CT is biosynthesized and secreted by gonadotrophs in the anterior pituitary gland [15,21]. In addition, there are also reports of the presence of SCT-like immunoreactive peptide in the mammalian pituitaries including rodents and humans [15,38,39]. Moreover, both SCT and HCT (or rat CT) have been shown to interact with the same CTR, and SCT is generally found to be more potent than HCT in most biological assays [40][41][42][43].…”

Calcitonin Induces IL-6 Production via Both PKA and PKC Pathways in the Pituitary Folliculo-Stellate Cell Line

“…On the other hand, the mRNA of the C1b subtype was not detected in either mouse pituitary or TtT/GF cells. C1b is considered to be a receptor for endogenous sCT-like peptides (47)(48)(49). The physiological plasma level of CT in rats is approximately 60 pg/ml (0.02 nm) to 480 pg/ml (0.1 nm) (50).…”